Peptide/lipid complex formation by co-lyophilization

ABSTRACT

The invention relates to the formation of peptide/lipid vesicles and complexes through the co-lyophilization of peptides, preferably that are able to adopt an amphipathic alphahelical conformation, and one or more lipids. A single solution which solubilizes both the peptides and lipids or two separate solutions may be lyophilized.

This is a continuation of application Ser. No. 09/642363. filed Aug. 21,2000, now U.S. Pat. No. 6,455,088 which is a continuation of applicationSer. No. 08/942,597, filed Oct. 2, 1997, now U.S. Pat. No. 6,287,590.

1. FIELD OF THE INVENTION

The invention relates to the formation of peptide/lipid vesicles andcomplexes through the co-lyophilization of peptides, preferably that areable to adopt an amphipathic alpha-helical conformation, and one or morelipids. A single solution which solubilizes both the peptides and lipidsor a two separate solutions may be lyophilized. The methods are used togenerate stable peptide/lipid vesicles and complexes including but notlimited to micellar, spherical and discoidal complexes in bulkpreparations and in smaller units, as may be suitable for dosages.

2. BACKGROUND OF THE INVENTION

Liposomes are vesicles composed of at least one lipid bilayer membraneenclosing an aqueous core. Generally, phospholipids comprise the lipidbilayer, but the bilayer may be composed of other lipids. The aqueoussolution within the liposome is referred to as the “captured volume.”

Liposomes have been developed as vehicles to deliver drugs, cosmetics,bioactive compounds among other applications. The lipid bilayerencapsulates the drug, cosmetic, bioactive compound, and the like,within the captured volume of the liposome and the drug is expelled fromthe liposome core when the lipid bilayer comes in contact with a cellsurface membrane. The liposome releases its contents to the cell bylipid exchange, fusion, endocytosis, or adsorption. Ostro et al., 1989,Am. J. Hosp. Pharm. 46:1576. Alternatively, the drug, cosmetic,bioactive compound and the like could be associated with or insertedinto the lipid bilayer membrane of the vesicle.

In addition to vesicles, lipid-containing complexes have been used todeliver agents in particle form. For instance, many researchers havefound it useful to prepare reconstituted lipoprotein-like particles orcomplexes which have similar size and density as high densitylipoprotein (HDL) particles. These reconstituted complexes usuallyconsist of purified apoproteins (usually apoprotein A-1) andphospholipids such as phosphatidylcholine. Sometimes unesterifiedcholesterol is included as well. The most common methods of preparingthese particles are (1) co-sonication of the constituents, either bybath sonication or with a probe sonicator, (2) spontaneous interactionof the protein constituent with preformed lipid vesicles, (3)detergent-mediated reconstitution followed by removal of the detergentby dialysis. Jonas, 1986, Meth. in Enzymol. 128:553–582; Lins et al.,1993, Biochimica et Biophysica Acta, 1151:137–142; Brouillette &Anantharamaiah, 1995, Biochimica et Biophysica Acta, 1256:103–129;Jonas, 1992, Structure & Function of Apoproteins, Chapter 8:217–250.Similar complexes have also been formed by substituting amphipathichelix-forming peptides for the apoprotein components. Unfortunately,each of these methods presents serious problems for the formation oflarge amounts of pure complexes on a reasonably cost-effective basis.Further, none of these publications disclose the co-lyophilization ofpeptides/or peptides analogues which are able to adopt an amphipathicalpha helical conformation and a lipid.

A range of technologies is known for producing lipid vesicles andcomplexes. Vesicles, or liposomes, have been produced using a variety ofprotocols, forming different types of vesicles. The various types ofliposomes include: multilamellar vesicles, small unilamellar vesicles,and large unilamellar vesicles.

Hydration of phospholipids (or other lipids) by aqueous solution canalso result in the dispersion of lipids and spontaneous formation ofmultimellar vesicles (“MLVs”). An MLV is a liposome with multiple lipidbilayers surrounding the central aqueous core. These types of liposomesare larger than small unilamellar vesicles (SUVs) and may be 350–400 nmin diameter. MLVs were originally prepared by solubilizing lipids inchloroform in a round-bottom flask and evaporating the chloroform untilthe lipid formed a thin layer on the wall of the flask. The aqueoussolution was added and the lipid layer was allowed to rehydrate.Vesicles formed as the flask is swirled or vortexed. Deamer et al.,1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York (citingBangham et al., 1965, J. Mol. Biol. 13:238). Johnson et al. subsequentlyreported that this method also generated single lamellar vesicles.Johnson et al., 1971, Biochim. Biophys. Acta 233:820.

A small unilamellar vesicle (SUV) is a liposome with a single lipidbilayer enclosing an aqueous core. Depending on the method employed togenerate the SUVs, they may range in size from 25–110 nm in diameter.The first SUVs were prepared by drying a phospholipid preparation inchloroform under nitrogen, adding the aqueous layer to produce a lipidconcentration in the millimolar range, and sonicating the solution at45° C. to clarity. Deamer et al., 1983, in Liposomes (Ostro, Ed.),Marcel Dekker, Inc. New York. SUVs prepared in this fashion yieldedliposomes in the range of 25–50 nm in diameter.

Another method of making SUVs is rapidly injecting an ethanol/lipidsolution into the aqueous solution to be encapsulated. Deamer et al.,1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York (citingBatzri et al., 1973, Biochim. Biophys. Acta 298:1015). SUVs produced bythis method range in size from 30–110 nm in diameter.

SUVs may also be produced by passing multilamellar vesicles through aFrench Press four times at 20,000 psi. The SUVs produced will range insize from 30–50 nm in diameter. Deamer et al., 1983, in Liposomes(Ostro, Ed.), Marcel Dekker, Inc. New York (citing Barenholz et al.,1979, FEBS Letters 99:210).

Multilamellar and unilamellar phospholipid vesicles can also be formedby extrusion of aqueous preparations of phospholipids at high pressurethrough small-pore membranes (Hope et al., 1996, Chemistry and Physicsof Lipids, 40:89–107)

A large unilamellar vesicle (LUV) is similar to SUVs in that they aresingle lipid bilayers surrounding the central aqueous core, but LUVs aremuch larger that SUVs. Depending on their constituent parts and themethod used to prepare them, LUVs may range in size from 50–1000 nm indiameter. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker,Inc. New York. LUVs are usually prepared using one of three methods:detergent dilution, reverse-phase evaporation, and infusion.

In the detergent dilution technique, detergent solutions such ascholate, deoxycholate, octyl glucoside, heptyl glucoside and TritonX-100 are used to form micelles from the lipid preparation. The solutionis then dialyzed to remove the detergent and results in the formation ofliposomes. Deamer et al., 1983, in Liposomes (Ostro, Ed.), MarcelDekker, Inc. New York. This method is time consuming and removal of thedetergent is generally incomplete. The presence of detergent in thefinal preparation may result in some toxicity of the liposomepreparation and/or modification of the physicochemical properties of theliposome preparation.

The reverse-phase evaporation technique solubilizes lipid inaqueous-nonpolar solutions, forming inverted micelles. The nonpolarsolvent is evaporated and the micelles aggregate to form LUVs. Thismethod generally requires a great deal of lipid.

The infusion method injects a lipid solubilized in a non-polar solutioninto the aqueous solution to be encapsulated. As the nonpolar solutionevaporates, lipids collect on the gas/aqueous interface. The lipidsheets form LUVs and oligolamellar liposomes as the gas bubbles throughthe aqueous solution. Liposomes are sized by filtration. Deamer et al.,1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York (citingDeamer et al., 1976, Biochim. Biophys. Acta 443:629 and Schieren et al.,1978, Biochim. Biophys. Acta 542:137). Infusion procedures require afairly high temperature for infusion and may have a relatively lowencapsulation efficiency. Deamer et al., 1983, in Liposomes (Ostro,Ed.), Marcel Dekker, Inc. New York

It is-has been a goal of liposome research to develop liposomepreparations that may be stored for long periods of time before use. Forexample, U.S. Pat. No. 4,229,360 to Schneider et al., discloses a methodof dehydrating liposomes by adding a hydrophilic compound to a colloidaldispersion of liposomes in an aqueous liquid and dehydrating thesolution, referably by lyophilization. Examples of hydrophilic compoundsare high molecular weight hydrophilic polymers or low molecular weightcompounds such as sucrose.

U.S. Pat. No. 4,411,894 to Shrank et al., discloses the use of highconcentrations of sucrose in sonicated preparations of liposomes. Theliposomes contain fat-soluble products in the captured volume, althoughthe preparations could be lyophilized, the method could not prevent theloss of a significant amount of the captured contents despite the highconcentration of sucrose.

Crowe et al., U.S. Pat. No. 4,857,319 disclosed the use of disaccharidessuch as sucrose, maltose, lactose and trehalose to stabilize liposomeswhen liposomes are freeze dried. The amount of disaccharide with respectto the lipid content of the component (w/w) is within 0.1:1 to 4:1.Crowe achieved greater success in preserving liposomal integrity usingthis method than that afforded by the method disclosed by Shrank in U.S.Pat. No. 4,441,894.

Janoff et al, U.S. Pat. No. 4,880,635 disclose a method for dehydratingliposomes in which liposomes were lyophilized in the presence ofprotective sugars such as trehalose and sucrose, preferably on both theinner and outer leaflets of the lipid bilayer. Sufficient water isretained in the method of Janoff et al. so that rehydration of the driedliposomes yields liposomes with substantial structural integrity.

However, there is a need in the art for a simple and cost effectivemethod of forming lyophilized peptide/lipid complexes which may be thenbe rehydrated. The method of the resent invention yields peptide/lipidmixtures in a stable, lyophilized powder which may be stored, used as apowder, or used after rehydration to form peptide/lipid complexes.

SUMMARY OF THE INVENTION

The invention is a method for preparing peptide orprotein-(phospho)lipid complexes or vesicles which may havecharacteristics similar to high density lipoprotein (HDL). The methodutilizes a solvent system in which at least one peptide is solubilizedin one solution, and at least one lipid is solubilized in anothersolution. The two solutions are selected such that they are misciblewith one another. The solutions are then combined, and the resultingsolution is lyophilized.

The method also may be practiced by a second type of solvent systemcomprising a solution into which both the protein or peptide and thelipid may be solubilized. This solution may be a single solution, or maybe a composite solution made by combining two or more solutions beforethe addition of peptides and lipids. Peptides and lipids are solubilizedin the solution or composite solution and the peptide/lipid solution isthen lyophilized.

Preferably, the peptides of the present invention are peptides which areable to adopt an amphipathic helical conformation. In one specificembodiment of the invention, the peptide is a lipid binding protein. Inother embodiment, peptide analogues of ApoA-I, ApoA-II, ApoA-IV, ApoC-I,ApoC-II, ApoC-III, ApoE, other apolipoprotein analogues and the like areutilized in place of or in combination with the peptides. In anotherspecific embodiment, the method is used to prepare ApoA1analogue/(phospho)lipid complexes similar to HDL. The ApoA1/lipidcomplexes are useful in treating disorders associated withdyslipoproteinemias including but not limited to hypercholesterolemia,hypertriglyceridemia, low HDL, and apolipoprotein A-1 deficiency, septicshock, for in vitro diagnostic assays as markers for HDL populations,and for use with imaging technology.

The method of the invention enables the preparation of peptide/lipidcomplexes for parenteral administration including but not limited tointravenous, intraperitoneal, subcutaneous, intramuscular, and bolusinjections to animals or humans. Further, the peptide/lipid complexescan also be formulated for oral, rectal, mucosal (e.g. oral cavity) ortopical administration to animals or humans, or for in vitroexperimentation.

The method may be used for large scale production of amphipathicpeptide/phospholipid complexes, lipid binding protein/phospholipidcomplexes, and/or ApoA1 peptide analogue/phospholipid complexes. Thelyophilized material may be prepared for bulk preparations, oralternatively, the mixed peptide/lipid solution may be apportioned insmaller containers (for example, single dose units) prior tolyophilization, and such smaller units may be prepared as sterile unitdosage forms.

The lyophilized powder prepared by the method of the invention can berehydrated into a particulate-free sterile solution immediately beforeinjection, or alternatively, the lyophilized powder can be formulatedinto an appropriate solid dosage form and administered directly.

The method may also be suitable for storage of compounds which may beotherwise unstable or insoluble in the absence of lipids.

The method may be used for the formulation of products for the treatmentor prevention of human diseases, including such applications asco-presentation of antigens in vaccines, treatment or prevention ofdyslipoproteinemias, including but not limited to hypercholesterolemia,hypertriglyercidemia, low HDL, and apolipoprotein A-1 deficiency,cardiovascular disease such as atherosclerosis, septic shock, orinfectious diseases.

The method may be used for the preparation of complexes that could beused as carriers for drugs, as vectors (to deliver drugs, DNA, genes),for example, to the liver or to extrahepatic cells, or as scavengers totrap toxin (e.g. pesticides, LPS, etc.).

3.1. DEFINITIONS

As used herein, a “solvent system” refers to one or more solvents whichare capable of solubilizing peptides and/or lipids and, if more thanone, which are miscible with one another.

As used herein, “peptide/lipid complexes” refers to an aggregation oflipid moieties and peptides forming particles within the size range ofhigh density lipoproteins (HDLs).

As used herein, “co-lyophilized” refers to the lyophilization,freeze-drying, or vacuum drying of more than one compound (e.g.,peptide, protein, lipid, phospholipid) in solution in the same vessel.For example, a lipid solution may be combined with a peptide solution inthe same vessel and the resulting combination of solutions islyophilized together, thereby lyophilizing the peptides and lipidssimultaneously.

As used herein “amphipathic peptide” or “amphipathic alpha helicalpeptides” means peptides which are able to adopt an amphipathic oramphipathic helical conformation, respectively. The amphipathic alphahelix is an often encountered secondary structural motif in biologicallyactive peptides and proteins. See Amphipathic helix motif: classes andproperties by Jere P. Segrest, Hans de Loof, Jan G. Dohlman, Christie G.Brouillette, and G. M. Anantharamaiah. PROTEINS: Structure Functions andGenetics 8:103–117 (1990). An amphipathic alpha helix is an alpha helixwith opposing polar and nonpolar faces oriented along the long axis ofthe helix. A specific distribution of charged residues is evident alongthe polar face. Amphipathic helices, as defined, are complementary forthe polar-nonpolar interface of hydrated bulk phospholipid; theselipid-associating domains have been postulated to interact with thephospholipid by partially immersing themselves at the interface betweenthe fatty acyl chains and the polar head groups. Jere P. Segrest. Febsletters 1976, 69 (1): 11–114.

The term l“peptide” and “protein” may be used interchangeably herein.Further, the peptide analogues of the invention can be peptides,proteins or non-peptides i.e., peptidomimetics. However, all theanalogues are preferably bioactive molecules.

The term “lipid” as used herein includes but is not limited to naturaland synthetic phospholipids. Further, the terms, “Lipid” and“phospholipid” may be used interchangeably herein.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Superose 6 chromatography of HDL prepared by densityultracentrifugation from 200 μl human serum.

FIG. 2 (bottom): Superose 6 chromatography of (DPPC: peptide 1)(PVLDLFRELLNELLEALKQKLK; SEQ ID NO:1) complexes prepared at a ratio of1:1 (w:w).

FIG. 2 (top): Superose 6 chromatography of (DPPC: peptide 1) complexesprepared at a ratio of 2:1 (w:w).

FIG. 3 (bottom): Superose 6 chromatography of (DPPC: peptide 1)complexes prepared at a ratio of 3:1 (w:w).

FIG. 3 (top): Superose 6 chromatography of (DPPC: peptide 1) complexesprepared at a ratio of 4:1 (w:w).

FIG. 4 (bottom): Superose 6 chromatography of (DPPC: peptide 1)complexes prepared at a ratio of 5:1 (w:w).

FIG. 4 (top): Superose 6 chromatography of (DPPC: peptide 1) complexesprepared at a ratio of 7.5:1 (w:w).

FIG. 5: Superose 6 chromatography of (DPPC: peptide 1) complexesprepared at a ratio of 10:1 (w:w).

FIG. 6: Superose 6 chromatography of ¹⁴C-labeled peptide 1 complexes atRi=3:1.

FIG. 7: Superose 6 chromatography of ¹⁴C-labeled peptide 1 complexes atRi=4:1.

FIG. 8: Superose 6 chromatography of ¹⁴C-labeled peptide 1 complexes atRi=5:1.

5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The amphipathic alpha helical peptides or proteins, lipid bindingproteins, ApoA-I agonist peptides, apoprotein analogues, and the like,which are useful in the present invention, can be synthesized ormanufactured using any technique known in the art. Stable preparationsof peptides which have a long shelf life may be made by lyophilizing thepeptides—either to prepare bulk for reformulation, or to prepareindividual aliquots or dosage units which can be reconstituted byrehydration with sterile water or an appropriate sterile bufferedsolution prior to administration to a subject.

To the inventor's knowledge, this invention is the first instance of amethod for co-lyophilizing an amphipathic alpha helical peptide orpeptide analogue with a lipid to form a mixture that can bereconstituted into a sterile peptide/lipid complex.

In certain embodiments, it may be preferred to formulate and administerthe ApoA-I analog(s) including but not limited to ApoA-I agonists, in apeptide-lipid complex. This approach has several advantages since thecomplex should have an increased half-life in the circulation,particularly when the complex has a similar size and density to the HDLclass of proteins, especially the pre-beta HDL populations. The HDLclass of lipoproteins can be divided into a number of subclasses basedon such characteristics as size, density and electrophoretic mobility.Some examples, in order of increasing size are micellar pre-beta HDL ofdiameter 50 to 60 Angstroms, discoidal HDL of intermediate size i.e.,with a mass of 65 kDa (about 70 Angstroms), spherical HDL₃ or HDL₂ ofdiameter 90 to 120 Angstroms. (J. Kane, 1996 in V. Fuster, R. Ross andE. Topol [eds.] Atherosclerosis and Coronary Artery Disease, p. 99; A.Tall and J. Breslow, ibid., p. 106; Barrans et al., Biochemica etBiophysica Acta 1300, p. 73–85; and Fielding et al., 1995, J. Lipid Res36, p,. 211–228). However, peptide/lipid complexes of smaller or largersize than HDL may also be formed by the invention.

The peptide-lipid complexes of the present invention can conveniently beprepared as stable preparations, having a long shelf life, by theco-lyophilization procedure described below. The lyophilizedpeptide-lipid complexes can be used to prepare bulk drug material forpharmaceutical reformulation, or to prepare individual aliquots ordosage units which can be reconstituted by rehydration with sterilewater or an appropriate buffered solution prior to administration to asubject.

The applicants have developed a simple method for preparing peptide orprotein-(phospho)lipid complexes which have characteristics similar toHDL. This method can be used to prepare the ApoA-I peptide-lipidcomplexes, and has the following advantages: (1) Most or all of theincluded ingredients are used to form the designed complexes, thusavoiding waste of starting material which is common to the othermethods. (2) Lyophilized compounds are formed which are very stableduring storage. The resulting complexes may be reconstituted immediatelybefore use. (3) The resulting complexes usually do not require furtherpurification after formation or before use. (4) Toxic compounds,including detergents such as cholate, are avoided. Moreover, theproduction method can be easily scaled up and is suitable for GMPmanufacture (i.e., in an endotoxin-free environment).

In accordance with the preferred method, the peptide and lipid arecombined in a solvent system which co-solubilizes each ingredient. Tothis end, solvent pairs must be carefully selected to ensureco-solubility of both the amphipathic peptide and the hydrophobic lipid.In one embodiment, the protein(s) or peptide(s) to be incorporated intothe particles can be dissolved in an aqueous or organic solvent ormixture of solvents (solvent 1). The (phospho)lipid component isdissolved in an aqueous or organic solvent or mixture of solvents(solvent 2) which is miscible with solvent 1, and the two solutions arecombined. Alternatively, the (phospho)lipid component is dissolveddirectly in the peptide (protein) solution. Alternatively, the peptideand lipid can be incorporated into a co-solvent system, i.e., a mixtureof the miscible solvents. Depending on the lipid binding properties ofthe peptide or protein, those skilled in the art will recognize thatenhanced or even complete solubilization (and/or enhanced mixing) may benecessary prior to lyophilization; thus, the solvents can be chosenaccordingly.

A suitable proportion of peptide (protein) to lipids is first determinedempirically so that the resulting complexes possess the appropriatephysical and chemical properties, usually but not always meaning similarin size to HDL₂ or HD₃. The lipid to protein/peptide molar ratio shouldbe in the range of about 2 to about 200, and preferably 5 to 50depending on the desired type of complexes. Examples of such sizeclasses of peptide/lipid or protein/lipid complexes include, but are notlimited to, micellar or discoidal particles (usually smaller than HDL₃or HDL₂), spherical particles of similar size to HDL₂ or HDL₃ and largercomplexes which are larger than HDL₂. The HDLs used by us as a standardduring chromatography (FIG. 1) are mainly spherical mature HDL₂. Pre-β1HDL are micellar complexes of apolipoprotein and few molecules ofphospholipids. Pre-β2 HDL are discoidal complexes of apolipoprotein andmolecules of phospholipids. The more lipids (triglycerides, cholesterol,phospholipids) are incorporated the bigger will become the HDL and itsshape is modified. (Pre-β1 HDL (micellar complex)

Pre-β2 HDL (discoidal complex))

HDL3 (spherical complex)

HDL2 (spherical complex).

Once the solvent is chosen and the peptide and lipid have beenincorporated, the resulting mixture is frozen and lyophilized todryness. Sometimes an additional solvent is added to the mixture tofacilitate lyophilization. This lyophilized product can be stored forlong periods and will remain stable.

In the working examples describe infra, the peptide 1PVLDLFRELLNELLEALKQKLK (SEQ ID NO:1) and (phospho)lipid were dissolvedseparately in methanol, combined, then mixed with xylene beforelyophilization. The peptide and lipid can both be added to a mixture ofthe two solvents. Alternatively, a solution of the peptide dissolved inmethanol can be mixed with a solution of lipid dissolved in xylene. Careshould be taken to avoid salting out the peptide. The resulting solutioncontaining the peptide and lipid co-solubilized in methanol/xylene islyophilized to form a powder.

The lyophilized product can be reconstituted in order to obtain asolution or suspension of the peptide-lipid complex. To this end, thelyophilized powder is rehydrated with an aqueous solution to a suitablevolume (often about 5 mg peptide/ml which is convenient for intravenousinjection). In a preferred embodiment the lyophilized powder isrehydrated with phosphate buffered saline or a physiological salinesolution. The mixture may have to be agitated or vortexed to facilitaterehydration, and in most cases, the reconstitution step should beconducted at a temperature equal to or greater than the phase transitiontemperature (Tm) of the lipid component of the complexes. Withinminutes, a solution of reconstituted lipid-protein complexes (a clearsolution when complexes are small) results.

An aliquot of the resulting reconstituted preparation can becharacterized to confirm that the complexes in the preparation have thedesired size distribution, e.g., the size distribution of HDL. Gelfiltration chromatography can be used to this end. In the workingexamples described infra, a Pharmacia Superose 6 FPLC gel filtrationchromatography system was used. The eluant used contains 150 mM NaCl indeionized water. A typical sample volume is 20 to 200 microliters ofcomplexes containing 5 mg peptide/ml. The column flow rate is 0.5ml/min. A series of proteins of known molecular weight and Stokes'diameter as well as human HDL are used as standards to calibrate thecolumn. The proteins and lipoprotein complexes are monitored byabsorbance or scattering of light of wavelength 254 or 280 nm.

The solvents that may be used according to the method of the presentinvention include but are not limited to nonpolar, polar, aprotic, andprotic organic solvents and the like such as ethanol, methanol,cyclohexane, 1-butanol, isopropyl alcohol, xylene, THF, ether, methylenechloride benzene and chloroform. The invention also includes the use ofsolvent mixtures as well as single solvents. Further, prior to usewithin the present methods the organic solvents maybe dried to removewater; however, hydrated solvents or water may be used with certainlipids, peptides or proteins. In other words, water may be a suitablesolvent, or hydrated solvents or organic solvent/water mixtures may beused, however, if water is used it must be detergent free. As mentionedabove, the solvents are preferably of the purest quality (in order toavoid concentrating impurities after lyophilization), and the solventsshould be salt free and free of particulates. However, the solvents neednot be sterile as the resulting product can be sterilized before, duringor after lyophilization, in accordance with known techniques in thepharmaceutical art, such as those described in Remington'sPharmaceutical Sciences, 16th and 18th Eds., Mack Publishing Co.,Easton, Pa. (1980 and 1990), herein incorporated by reference in itsentirety, and in the United States Pharmacopeia/National Formulary(USP/NF) XVII, herein incorporated by reference in its entirety.

The lipids which may be used according to the method of the presentcomposition include but are not limited to natural and synthesized(synthetic) lipids and phospholipids including small alkyl chainphospholipids, egg phosphatidylcholine, soybean phosphatidylcholine,dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine,distearoylphosphatidylcholine1-myristoyl-2-palmitoylphosphatidylcholine,1-palmitoyl-2-myristoylphosphatidylcholine,1-palmitoyl-2-stearoylphosphatidylcholine,1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholinedioleophosphatidylethanolamine, dilauroylphosphatidylglycerolphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,phosphatidylinositol, sphingomyelin sphingolipids, phosphatidylglycerol,diphosphatidylglycerol diinyristoylphosphatidylglycerol,dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol,dioleoylphosphatidylglycerol, dimyristoylphosphatidic aciddipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine,dipalmitoylphosphatidylserine, brain phosphatidylserine, brainsphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin,phosphatidic acid, galactocerebroside, gangliosides, cerebrosides,dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride,aminophenylglycoside, 3-cholesteryl-6′-(glycosylthio)hexyl etherglycolipids, and cholesterol and its derivatives.

The peptides that are suitable for use with the present inventioninclude, but are not limited to, those described in Ser. No. 08/940,095,filed Sep. 29. 1997, now U.S. Pat. No. 6,004,925; Ser. No. 08/940,096,filed Sep. 29, 1997, now Pat. No. 6,046,166; and Ser. No. 08/940,093,filed Sep. 29, 1997, now Pat. No. 6,037,323, each of which is herebyincorporated by reference in its entirety.

It is preferred, although not necessary in every case, that precipitatesshould be solubilized or removed prior to mixing or stirring the lipidand peptide solutions or prior to lyophilization.

The method may be used for large scale production of peptide/lipidcomplexes, amphipathic peptide/(phospho)lipid complexes, lipid bindingprotein/(phospho)lipid complexes, and/or ApoA1 peptideanalogue/(phospho)lipid complexes. The lyophilized material may beprepared for bulk preparations, or alternatively, the mixedpeptide/lipid solution may be apportioned in smaller containers (forexample, single dose 10 units) prior to lyophilization, and such smallerunits may be prepared as sterile single dosage forms.

The vacuum dried compositions of the present invention may be providedin single dose or multiple dose container forms by aseptically fillingsuitable containers with the sterile pre-vacuum dried solution to aprescribed content; preparing the desired vacuum dried compositions; andthen hermetically sealing the single dose or multiple dose container. Itis intended that these filled containers will allow rapid dissolution ofthe dried composition upon reconstitution with appropriate sterilediluents in situ giving an appropriate sterile solution of desiredconcentration for administration. As used herein, the term “suitablecontainers” means a-container capable of maintaining a sterileenvironment, such as a vial, capable of delivering a vacuum driedproduct hermetically sealed by a stopper means. Additionally, suitablecontainers implies appropriateness of size, considering the volume ofsolution to be held upon reconstitution of the vacuum dried composition;and appropriateness of container material, generally Type I glass. Thestopper means employed, e.g., sterile rubber closures or the equivalent,should be understood to be that which provides the aforementioned seal,but which also allows entry for the purpose of the introduction of adiluent, e.g., sterile Water for Injection, USP, Normal Saline, USP, or5% Dextrose in Water, USP, for the reconstitution of the desiredsolution. These and other aspects of the suitability of containers forpharmaceutical products such as those of the instant invention are wellknown to those skilled in the practice of pharmaceutical arts. Inspecific embodiments, sizes of product unit dosages may be in a range ofabout 10 mg to 2 g of peptide preferably in the range of about 100 mg to1 g and at a concentration after reconstitution of about 1 to 50 mg/ml,preferably about 2 to 25 mg/ml.

The method of the invention enables the preparation of protein orpeptide/lipid complexes for parenteral administration includingintravenous, intraperitoneal, subcutaneous, intramuscular and bolusinjections to animals or humans, or for oral, rectal, mucosal (e.g. oralcavity) or topical administration to animals or humans, or for in vitroexperimentation.

The lyophilized powder prepared by the method of the invention can berehydrated immediately before injection, or alternatively, thelyophilized powder can be administered directly. The lyophilized powderincludes, but is not limited to lipid and peptides that are able to formcomplexes in the form of vesicles, liposomes, particles includingspherical or discoidal particles, micelles and the like. In order toreconstitute or rehydrate the lyophilized powder a solution is chosendepending upon the desired end use. For pharmaceutical use any sterilesolution may be used. Further, buffered solutions are preferred forcertain uses and these include but are not limited to phosphate,citrate, tris, baribital, acetate, glycine-HCl, succinate, cacodylate,boric acid-borax, ammediol and carbonate.

The lyophilized powder of the present invention may be formed using anymethod of lyophilization known in the art, including, but not limitedto, freeze-drying in which the peptide/lipid-containing solution issubjected to freezing followed by reduced pressure evaporation.

The method may also be suitable for storage of compounds which may beotherwise unstable or insoluble in the absence of lipids.

The method may be used for the formulation of products for the treatmentor prevention of human diseases, including such applications asco-presentation of antigens in vaccines, treatment or prevention ofdyslipoproteinemias including but not limited to hypercholesterolemia,hypertriglyceridemia, low HDL, and apolipoprotein A-1 deficiency,cardiovascular disease such as atherosclerosis, septic shock, orinfectious diseases.

The method may be used for the preparation of complexes that could beused as carriers for drugs, as vectors (to deliver drugs, DNA, genes),for example, to the liver or to extrahepatic cells, or as scavengers totrap toxin (e.g. pesticides, LPS, etc.). Alternatively, the method maybe used to prepare complexes for in vitro assay systems, or for use inimaging technology.

In specific embodiments, the method may be used for the preparation ofApoA-I analogue (including but not limited to agonists) complexes whichmay be used in in vitro diagnostic assays and as markers for HDLpopulations and subpopulations. In other specific embodiments, ApoA-Iagonist complexes may be used for immunoassays or for imaging technology(e.g., CAT scans, MRI scans).

The following examples are intended to be illustrative of the presentinvention and should not be construed, in any way, to be a limitationthereof.

6. EXAMPLE Preparation of Peptide-lipid Complex by Co-LyophilizationApproach

The following protocol was utilized to prepare peptide-lipid complexes.

Peptide 1 (PVLDLFRELLNELLEALKQKLK; SEQ ID NO:1) (22.4 mg) was dissolvedin methanol at a concentration of 3.5 mg/ml by incubation for severalminutes and mixing by vortex intermittently. To this solution was addeddipalmitoylphosphatidylcholine (DPPC) in methanol (100 mg/ml stocksolution) such that the final ratio of DPPC/peptide was 2.5:1(weight/weight). This solution was mixed by vortexing. Xylene was addedto the solution to a final concentration of 36%. Aliquots of theresulting solution were removed for later analysis by gel filtrationchromatography. The solutions were frozen in liquid nitrogen andlyophilized to dryness by vacuum. An aliquot containing 20 mg peptide 1(SEQ ID NO:1) and 50 mg DPPC was rehydrated in sterile saline solution(0.9% NaCl), mixed, and heated to 41° C. for several minutes until aclear solution of reconstituted peptide/phospholipid complexes resulted.

6.1. EXAMPLE Gel Filtration and Phospholipid Utilization 6.1.1.Materials and Methods

For the purpose of testing conditions for the preparation of complexesit is often convenient to prepare small amounts of complexes forcharacterization. These preparations contained one mg of peptide andwere prepared as follows: One mg of peptide 1 (SEQ ID NO: 1) wasdissolved in 250 μl HPLC grade methanol (Perkin Elmer) in a 1.0 ml clearglass vial with cap (Waters #WAT025054). Dissolving of the peptide wasaided by occasional vortexing over a period of 10 minutes at roomtemperature. After this time a small amount of undissolved particulatematter could still be seen but this did not adversely affect theresults. To this mixture an aliquot containing either 1, 2, 3, 4, 5,7.5, 10 or 15 mg DPPC (Avanti Polar Lipids, 99% Purity, product #850355)from a 100 mg/ml stock solution in methanol was added. The volume of themixture was brought to 400 μl by addition of methanol and the mixturewas further vortexed intermittently for a period of 10 minutes at roomtemperature. At this time, very little undissolved material could beseen in the tubes. To each tube 200 μl of xylene (Sigma-Aldrich 99%pure, HPLC-grade) was added and the tubes were vortexed for 10 secondseach. Two small holes were punched into the tops of each tube with a 20gauge syringe needle, the tubes were frozen for 15 seconds each inliquid nitrogen, and the tubes were lyophilized overnight under vacuum.To each tube 200 ml of 0.9% NaCl solution was added. The tubes werevortexed for 20 seconds each. At this time the solutions in the tubeswere milky in appearance. The tubes were then incubated in a water bathfor 30 minutes at 41° C. The solutions in all of the tubes became clear(i.e., similar to water in appearance) except for the tube containing 15mg DPPC, which remained milky.

In order to determine if all of the phospholipids that were used in thecomplex preparations actually appeared in the column fractionscorresponding to the chromatogram absorbance peaks, the column eluatefrom reconstituted peptide/lipid complexes was collected in one or twoml fractions and the fractions were assayed enzymatically forphospholipid content with the BioMerieux Phospholipides Enzymatique PAP150 kit (#61491) according to the instructions supplied by themanufacturer.

The preparations of complexes may also be done on a larger scale. Anexample of one such preparation is reported above. These complexes wereused for in vivo experiments.

6.2. Results of Complex Characterization

FIG. 1: Superose 6 chromatography of mature HDL prepared by densityultracentrifugation from 200 μl human serum. Chromatograph showsabsorbance at 254 nm. Elution volume=14.8 ml, corresponding to a Stokes'diameter of 108 Angstroms (See Table 1).

FIG. 2 (bottom): Superose 6 chromatography of DPPC:peptide 1 complexesprepared at a ratio of incubation (Ri, defined as the ratio of totalphospholipid to total peptide in starting mixture) of 1:1 (w:w) asdescribed above (small scale preparation). Elution volumes of absorbancepeaks=16.2 mls and 18.1 ml corresponding to particles of Stokes'diameters 74 and 82 Angstroms, which are smaller than HDL. 87% of thephospholipid applied to the column was recovered in the fractionscontaining the absorbance peaks (See Table 1).

FIG. 2 (top): Superose 6 chromatography of DPPC:peptide 1 complexesprepared at an Ri of 2:1 (w:w) as described above. Elution volume ofabsorbance peak 16.4 ml, (77 Angstroms), corresponding to particlessmaller than HDL. 70% of the phospholipid applied to the column wasrecovered in the fractions containing the absorbance peak (See Table 1).

FIG. 3 (bottom): Superose 6 chromatography of DPPC:peptide 1 complexesprepared at an Ri of 3:1 (w:w) as described above. Elution volume ofabsorbance peak=16.0 ml, (80 Angstroms) corresponding to particlessmaller than HDL. 79% of the phospholipid applied to the column wasrecovered in the fractions containing the absorbance peak (See Table 1).

FIG. 3 (top): Superose 6 chromatography of DPPC:peptide 1 complexesprepared at an Ri of 4:1 (w:w) as described above. Elution volume of theabsorbance peak=15.7 ml, (90 Angstroms), corresponding to particlessmaller than HDL. 106% of the phospholipid applied to the column wasrecovered in the fractions containing the absorbance peak (See Table 1).

FIG. 4 (bottom): Superose 6 chromatography of DPPC:peptide 1 complexesprepared at an Ri of 5:1 (w:w) as described above. Elution volume of theabsorbance peak=15.1 ml, (104 Angstroms), corresponding to particlessmaller than HDL. 103% of the phospholipid applied to the column wasrecovered in the fractions containing the absorbance peak (See Table 1).

FIG. 4 (top): Superose 6 chromatography of DPPC:peptide 1 complexesprepared at an Ri of 7.5:1 (w:w) as described above. Elution volume ofthe absorbance peak=13.6 ml, (134 Angstroms) corresponding to particleslarger than HDL. 92% of the phospholipid applied to the column wasrecovered in the fractions containing the absorbance peaks (See Table1).

FIG. 5: Superose 6 chromatography of DPPC:peptide 1 complexes preparedat a ratio of 10:1 (w:w) as described above. Elution volume ofabsorbance peak=13.4 ml, (138 Angstroms), again corresponding toparticles larger than HDL. 103% of the phospholipid applied to thecolumn was recovered in the fractions containing the absorbance peaks(See Table 1).

The sample containing complexes with 15:1 DPPC:peptide 1 (w:w) was notsubjected to Superose 6 chromatography because it was turbid, suggestingthe presence of large particles.

For each of the above experiments, no significant phospholipid wasobserved in any fraction other than those containing material elutingwith the absorbance peaks (See FIGS. 2–8). This suggests that virtuallyall of the phospholipids (within experimental error of the assay) wereincorporated into the complexes. The experiment demonstrates that byvarying the initial ratio of phospholipids to peptides, homogeneouscomplexes of various sizes (smaller or larger than HDL) can be formed.

6.3. Characterization of Complexes using ¹⁴C-labeled Peptide 1

Peptide-phospholipid complexes containing ¹⁴C-labeled peptide 1(specific activity 159,000 DPM/mg peptide by weight, assuming 50%peptide content) were prepared by co-lyophilization as described above.The preparations each contained 1 mg peptide and 3, 4 or 5 mg DPPC byweight. After reconstituting the complexes in 200 μl 0.9% NaCl, 20 μl(100 μg) of the complexes were applied to a Pharmacia Superose 6 columnusing 0.9% NaCl as the liquid phase at a flow rate of 0.5 ml/min. Aftera 5 ml delay (column void volume=7.7 ml) 1 ml fractions were collected.Aliquots containing 20 μl of the fractions were assayed for phospholipidcontent using the BioMerieux enzymatic assay. The remainder of eachfraction was counted for 3 minutes in a Wallach 1410 liquidscintillation counter (Pharmacia) using the Easy Count program. Theresults of these analyses are shown in FIGS. 6–8. It can be seen thatthe vast majority of both phospholipid and peptide are recoveredtogether in a few fractions with peaks at approximately 16, 16, and 15ml for complexes prepared at 3:1, 4:1 and 5:1 DPPC:peptide ratios,respectively. The UV absorbance profiles for these samples indicate thatthe complexes elute from the column at volumes 15.1, 14.7 and 14.4 mlfor complexes prepared at 3:1, 4:1 and 5:1 DPPC:peptide ratios,respectively (the dead volume of tubing between the fraction collectorand UV flow cell is 1.3 ml, which explains a slight discrepancy betweenthe elution volumes as measured by radioactivity/phospholipid assay andUV absorbance). The elution volumes correspond to Stoke's diameters of106, 114, and 120 Angstroms for the 3:1, 4:1 and 5:1 Ri complexes,respectively.

TABLE 1 Relative % of Applied DPPC:Peptide 1 Size of Phospholipid inratio Elution Volume Particles* Absorbance Peak HDL 14.8 — — 1:1 16.2and 18.1 Smaller 87% 2:1 16.4 Smaller 70% 3:1 16.0 Smaller 79% 4:1 15.7Smaller 106%  5:1 15.1 Smaller 103%  7.5:1   13.6 Larger 92% 10:1  13.4Larger 103%  15:1  ND** ND ND *Relative to size of HDL particles **ND,not done

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

1. A process of preparing a lyophilized peptide/lipid product consistingessentially of co-lyophilizing a peptide consisting of the amino acidsequence of SEQ ID NO.:1, and one or more lipids in a solvent system toform a peptide/lipid product, wherein said product is suitable forrehydration to form peptide/lipid complexes, and wherein said solvent isan organic solvent or an aqueous/organic solvent mixture.